Chikungunya Virus Replication and Pathogenesis

基孔肯雅病毒复制和发病机制

基本信息

批准号：
9234459
负责人：
TERENCE S. DERMODY
金额：
$ 74.08万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9234459
关键词：
Acute Acute Disease Aedes Affect Alphavirus Americas Anabolism Antiviral Agents Arboviruses Arthritis Attenuated Binding Biochemical Biological Assay Biology Caribbean region Cell Culture Techniques Cell Line Cell membrane Cell-Matrix Junction Cells Chikungunya virus Chronic Chronic Disease Chronic Phase of Disease Clathrin Coat Protein Complex I Collaborations Colorado Culicidae Cytosol Defect Development Diagnosis Diamond Disease Endocytosis Endoplasmic Reticulum Endosomes Engineering Enzymes Epidemic Exanthema Fever Future GBF1 gene Gene Targeting Glycoproteins Glycosaminoglycans Golgi Apparatus Health Human Immune response Immunology Individual Infection Infiltration Inflammatory Inflammatory Response Injury Integration Host Factors Knockout Mice Knowledge Laboratories Leukocytes Link Mammalian Cell Mediating Mediator of activation protein Membrane Membrane Fusion Membrane Glycoproteins MicroRNAs Molecular Mus Musculoskeletal Mutagenesis North America Pathogenesis Pathogenicity Pathologic Pathology Pathway interactions Polyarthralgias Polysaccharides Property RNA Viruses Replicon Research Role Seeds Site Small Interfering RNA Structure Surface Testing Therapeutic Tissues Transport Vesicles Tropism Universities Vaccines Vesicle Transport Pathway Viral Viral Pathogenesis Virion Virulence Virulent Virus Virus Diseases Virus Receptors Virus Replication Washington Work base cell type cellular targeting chemokine cytokine experimental study genomic RNA improved insight mouse model mutant novel therapeutics public health relevance screening uptake viral RNA virus infection mechanism virus pathogenesis

项目摘要

DESCRIPTION (provided by applicant): Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes massive epidemics of a debilitating musculoskeletal inflammatory disease. There are currently no approved CHIKV-specific vaccines or antiviral agents. CHIKV initiates infection after the E2 glycoprotein binds to glycosaminoglycans (GAGs) on the surface of host cells and promotes internalization by clathrin-dependent uptake into the endocytic pathway. Both attenuated and virulent CHIKV strains bind GAGs, and GAG-binding efficiency influences virulence, including in a mouse model of CHIKV disease. However, the molecular basis of CHIKV-GAG interactions is unknown, which precludes a complete understanding of the function of GAGs in CHIKV pathogenesis. Moreover, the host factors that promote CHIKV replication, the precise cellular targets for CHIKV infection in the host, and links between host CHIKV replication factors and disease pathogenesis also remain unclear. The proposed research combines the expertise of the laboratories of Terence Dermody, Michael Diamond, and Thomas Morrison in virus-receptor interactions, RNA virus replication, viral immunology, and mouse models of viral disease to enhance knowledge of CHIKV replication and pathogenesis. Three integrated and interactive specific aims are proposed. In Specific Aim 1, the mechanisms and pathological significance of CHIKV binding to GAGs will be determined. Specific GAG subtypes bound by CHIKV will be defined using glycan-array screening and genetically altered cell lines with defects in GAG biosynthesis, and sequences in E2 required for binding to GAGs will be defined using structure-guided mutagenesis. The function of GAG binding in acute and chronic CHIKV disease will be determined using mutant CHIKV strains with reduced or abolished GAG-binding capacity and GAG-knockout mice. In Specific Aim 2, the function in CHIKV infection of COPI coatomer subunits and regulatory factors, which were recently identified in a small-interfering RNA screen, will be elucidated. Cells with diminished COPI transport activity will be infected with CHIKV and tested for formation of viral replication compartments, synthesis of viral RNA, and assembly and release of viral progeny. The function of COPI coatomer ARCN1 and regulatory factor GBF1 in CHIKV pathogenesis will be determined using newly established gene-targeted mice. In Specific Aim 3, cell types in the mammalian host that contribute to CHIKV pathogenesis and persistence will be identified. CHIKV strains engineered to contain tissue-specific microRNA seed sequences will be tested for acute and chronic disease in mice and elaboration of chemokines and cytokines, infiltration of musculoskeletal tissues with inflammatory leukocytes, and development of humoral immune responses. Overall, studies in this collaborative proposal will enhance our understanding of mechanisms by which CHIKV binds to GAGs, determine the function of COPI transport in CHIKV infection and pathogenesis, and define specific cells in the host targeted by CHIKV to produce disease. Knowledge gained from the proposed research may illuminate new targets for anti-CHIKV therapeutics.

描述（由适用提供）：Chikungunya病毒（CHIKV）是一种蚊子传播的α病毒，会引起大量衰弱的肌肉骨骼炎症性疾病。目前尚无认可的CHIKV特异性疫苗或抗病毒剂。 CHIKV启动E2糖蛋白后的感染与宿主细胞表面上的糖胺聚糖（GAGS）结合，并通过网格蛋白依赖性摄取到内吞途径中促进内在化。减毒和有毒的CHIKV菌株都结合了堵嘴，结合效率会影响病毒，包括在Chikv疾病的小鼠模型中。然而，Chikv-Gag相互作用的分子基础尚不清楚，该相互作用规定了对Chikv发病机理中GAG的功能的完全理解。此外，促进CHIKV复制的宿主因子，宿主中CHIKV感染的精确细胞靶标，以及宿主CHIKV复制因子与疾病发病机理之间的联系也尚不清楚。拟议的研究结合了Terence Dermody，Michael Diamond和Thomas Morrison实验室的专业知识，在病毒 - 受体相互作用，RNA病毒复制，病毒免疫学和病毒疾病的小鼠模型中，以增强CHIKV复制和致病性的知识。提出了三个集成和交互式特定的目标。在特定的目标1中，将确定CHIKV结合与GAGS的机制和病理意义。由CHIKV结合的特异性GAG亚型将使用聚糖阵列筛选和遗传改变的细胞系定义，并在GAG生物合成中缺陷，并且将使用结构引导的诱变来定义与GAG结合的E2中所需的E2序列。使用突变体CHIKV菌株（急性和慢性CHIKV疾病）在急性和慢性CHIKV疾病中的功能，具有降低或废除的GAG结合能力和GAG-KNOCKOUT小鼠的功能。在特定的目标2中，将阐明COPI座椅亚基和调节因子的CHIKV感染的功能，这些功能将在小型裂缝RNA筛选中鉴定出来。 COPI转运活性降低的细胞将被CHIKV感染，并测试形成病毒复制室的形成，病毒RNA的合成以及病毒后代的组装和释放。 COPI Coater ARCN1和调节因子GBF1在CHIKV发病机理中的功能将使用新建立的基因靶向小鼠确定。在特定的目标3中，将鉴定出有助于CHIKV发病机理和持久性的哺乳动物宿主中的细胞类型。 CHIKV菌株设计为包含组织特异性的微洋种子序列，将在小鼠中测试急性和慢性疾病，并阐述趋化因子和细胞因子，具有炎性白细胞的肌肉骨骼组织的浸润，并发育出色的免疫反应。总体而言，该协作提案中的研究将增强我们对CHIKV与插科打结合的机制的理解，确定COPI转运在CHIKV感染和发病机理中的功能，并定义CHIKV靶向产生疾病的宿主中的特定细胞。从拟议的研究中获得的知识可能会阐明抗chikv疗法的新目标。